Modulation of inflammation related to columnar epithelia

ABSTRACT

This invention provides pharmaceutical compositions containing lipoxin compounds and therapeutic uses for the compounds in treating or preventing a disease or condition associated with columnar epithelial inflammation. The invention also discloses methods for screening for compounds useful in preventing columnar epithelial inflammation.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/955,860 filedOct. 21, 1997 now abandoned which is a divisional of U.S. Ser. No.08/806,278 filed Feb. 25, 1997 now abandoned, which is a divisional ofU.S. Ser. No. 08/268,049 filed Jun. 29, 1994 now U.S. Pat. No. 5,650,435which is a which is a continuation-in-part application of U.S. Ser. No.08/084,311 filed Jun. 29, 1993 now abandoned, which in turn is acontinuation-in-part application of application U.S. Ser. No. 07/748,349now abandoned, filed Aug. 22, 1991, which in turn is acontinuation-in-part application of application U.S. Ser. No.07/677,388, filed Apr. 1, 1991 now abandoned. The contents of theaforementioned applications are hereby incorporated by reference.

GOVERNMENT SUPPORT

The work leading to this invention was supported by at least one grantfrom the U.S. Government. The U.S. Government, therefore, may beentitled to certain rights in the invention.

BACKGROUND

Columnar epithelia exist in the lungs, kidneys, bladder, bile ducts,pancreatic ducts, gall bladder, testicles, thyroid, trachea, intestine,stomach, and liver. In many disease states, polymorphonuclear leukocytes(PMN) migrate across these epithelia (Yardley J. H., et al. (1977). InThe Gastrointestinal Tract. Yardley and B. C. Morson, editors. Williamsand Wilkins Co., Baltimore. 57.) (Yardley, J. H. (1986). In RecentDevelopments in the Therapy of Inflammatory Bowel Disease. Proceedingsof a Symposium. Myerhoff Center for Digestive Disease at Johns Hopkins,Baltimore. 3-9.) This migration of PMN is an early event in themechanism of epithelial perturbation, which includes one or more of thefollowing events: abnormal fluid and electrolyte transport, specificepithelial barrier dysfunction, and ultimately mucosal breakdown. Theseperturbations lead to chronic and episodic inflammatory conditions.

Epithelial perturbations cause or contribute to many inflammatorydisease states including: gastritis, diverticulitis, cystic fibrosis,infectious colitis, bronchitis, asthma, Crohn's disease, nephritis,alveolitis, intestinal ulcers, idiopathic AIDS enteropathy,gastroenteritis, ischemic diseases, and glomerulonephritis. The efficacyof existing therapy for epithelial inflammation, such as methotrexate orcorticosteroids, is highly unsatisfactory, partially due to a hightoxicity which produces severe, adverse effects such as bone-weakeningand systemic immuno-suppression. (Physician's Desk Reference (41st ed.,1987) Medical Economics Co., Inc. 1103-1104.) Even under idealbioavailability conditions, the existing treatments fail tomechanistically target columnar epithelial inflammation.

New treatments for epithelial inflammation are needed.

SUMMARY OF INVENTION

This instant invention discloses new methods and compositions fortreating or preventing inflammation which is caused or contributed to bythe perturbation of columnar epithelia in a subject. The newpharmaceutical compositions comprise natural lipoxin A₄ or analogs oflipoxin A₄. And the new methods comprise administering to a subjecthaving a columnar epithelial inflammatory disease an effective,antiinflammatory amount of natural lipoxin A₄ or a lipoxin A₄ analog.

Natural lipoxin A₄ and analogs are thought to effect theirantiinhibitory activity by interfering with the interaction betweenpolymorphonuclear (PMN) cells and columnar epithelium. Migration of PMNis an early event in the mechanism of epithelial perturbation whichleads to mucosal breakdown, epithelial dysfunction, and chronicinflammatory conditions. As disclosed herein, prior exposure ofpolymorphonuclear leukocytes (PMN) to certain lipoxin compounds alterssubsequent PMN migration across the columnar epithelium, therebypreventing an inflammatory response. By inhibiting an early event in themechanism, LXA₄ effectively targets inflammation and inflammatoryresponses caused or contributed to by epithelial perturbation.

LXA₄, is a naturally-occuring tetraene-containing eicosanoids.Therefore, pharmaceutical compositions of LXA₄ or analogs thereof wouldexpected to be biocompatible. In addition, because LXA₄ and analogsthereof are highly potent in vivo, relatively small doses can beadministered to produce a therapeutic effect. In addition, naturallipoxins are subject to metabolic transformations in situ, that wouldfurther minimize any toxic, adverse effects, or adverse druginteractions. Alternatively, the instant invention discloses LXA₄analogs that are relatively resistant to in vivo degradation andtherefore, if shown to be safe, can be administered for a more prolongedtherapeutic effect. Lipophilic LXA₄ can be actively absorbed by columnarepithelial tissue.

For the reasons stated above, pharmaceutical compositions of naturalLXA₄ or analogs thereof provide a superior drug for treating columnarepithelial inflammatory diseases. Additional features and advantages ofthe invention will become more apparent from the following detaileddescription and claims.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to methods for treating or preventinginflammation or an inflammatory response caused or contributed to by theperturbation of a columnar epithelium. The term “columnar epithelium” isintended to mean one or more of the epithelia of the intestine, kidney,stomach, liver, thyroid, trachea, lung, gall bladder, urinary bladder,bile ducts, pancreatic ducts, liver, and testicles. A columnarepithelium performs three functions. First, it acts as a physicalbarrier. Second, it moves fluids, electrolytes, and nutrients in vectorsacross the epithelium. Third, it synthesizes and releases bioactivemolecules to influence other cell types.

An epithelial perturbation is a deleterious alteration of one or more ofthe following: the normal barrier function; the transportation offluids, electrolytes, or nutrients; or the synthesis or release ofbioactive molecules by the epithelial cells. The term “epithelialperturbation” is meant to include one or more of the following events:abnormal fluid and electrolyte transport, especially chloride ionsecretion, specific epithelial barrier dysfunction, and eventual mucosalbreakdown. These perturbations lead to chronic and episodic inflammatoryconditions.

This invention provides, in part, a method of screening for a compoundwhich attenuates abnormal fluid and electrolyte transportation, whichmay or may not be caused by activated inflammatory cells. This inventionalso provides a method of treating or preventing the symptoms ofabnormal fluid and electrolyte transportation, such as secretorydiarrhea by administering to a subject of an effective amount of anatural lipoxin or lipoxin analog, or combination thereof, to reduce orprevent an epithelial perturbation of fluid and electrolytetransportation.

Activation of one or more types of inflammatory cells can mediate thisinflammatory perturbation by inducing inflammatory cell action in theform of adhesion, migration, the release of bioactive molecules, or acombination thereof. Nonlimiting examples of inflammatory cells areleukocytes, which encompass polymorphonuclear leukocytes (PMN),eosinophils, T-lymphocytes, B-lymphocytes, natural killer cells, andmonocyte/macrophages. For example, migration of PMN across theepithelium of the intestine is an early event in the perturbationmechanism. The term “migration” is meant to include both the adhesion ofPMN to the epithelium and the complete traversion across the epitheliumto the other side. Under normal circumstances, PMN rarely adhere to theepithelial surface, and thus such adhesion is considered therate-lirniting step in the migratory process.

This invention provides, in part, a method of screening for a compoundwhich inhibits the activation of inflammatory cells, such as PMN, whichinteract with an epithelium. This method evaluates the anti-inflammatoryaction of an eicosanoid, such as a lipoxin, a lipoxin analog, or acombination thereof, based on the extent of its inhibition of PMNmigration in the basal-to-apical direction. This invention also providesa method of treating or preventing inflammation or the inflammatoryresponse caused or contributed to by activation of inflammatory cells.This method is the administration to a subject of an effective amount ofa lipoxin or lipoxin analog, or combination thereof, to reduce orprevent inflammatory cell activation and the consequent inflammatoryresponse.

This invention is based, in part, upon the finding that prior exposureof PMN to nanomolar concentrations of lipoxin A₄ (LXA₄) and certainlipoxin analogs modify subsequent PMN migration across an epithelialbarrier. The effect was found to be dependent on the direction of PMNtransepithelial migration: LXA₄ inhibited the number of migrating PMNcells in the basal-to-apical direction, but promoted the number ofmigrating PMN cells in the apical-to-basal direction. In a typicalembodiment of the screening method, the basal-to-apical inhibitionrepresented a decrease of 25%, and the apical-to-basal promotionrepresented an increase of 80%, after pretreatment of PMN with LXA₄ (10nM) for 15 minutes.

Inflammatory Diseases of Columnar Epithelia

Epithelial perturbations cause or contribute to inflammatory intestinaldisease states including: acute self-limited enterocolitis; viralinfections such as non-specific enteritis or specific viral enteritis;ulcerative colitis; Crohn's disease; diverticulitis; bacterialenterocolitis, such as salmonellosis, shigellosis, campylobacterenterocolitis, or yersinia enterocolitis; protozoan infections such asamebiasis; helminthic infection; and pseudomembraneous colitis.

Additional inflammatory intestinal diseases are duodenitis resultingcaused by infections, physical and chemical injuries, Celiac disease,allergic disease, immune disorders or stress ulcers; lymphocyticcolitis; collagenous colitis; diversion-related colitis; acuteself-limited colitis; microscopic colitis; solitary rectal ulcersyndrome; Behcet's disease; nonspecific ulcers of the colon; secondaryulcers of the colon; ischemic bowel disease; vasculitis; pepticduodenitis; peptic ulcer; bypass enteritis; ulcerative jejunoileitis; ornonspecific ulcers of the small intestine. Malabsorptive disordersinclude mucosal lesions associated with altered immune response such asidiopathic AIDS enteropathy, with viral or bacterial infections, or withmiscellaneous diseases such as mastocytosis or eosinophilicgastroenteritis.

Perturbations of the epithelia of the lung and trachea cause orcontribute to inflammatory lung diseases such as: cystic fibrosis,bronchiolitis, bronchitis, asthma, interstitial lung disease,eosinophilic pneumonias, tracheobronchitis, tracheoesophageal fistulas,and alveolitis.

Perturbations of the epithelium of the kidney cause or contribute todiseases such as: glomerulonephritis, nephritis, polycystic disease,ischemic disease, immune-complex-induced disease, immunopathogenicinjuries, pyelonephritis, and tubulointerstitial disease.

Perturbations of the epithelium of the stomach cause or contribute todiseases such as gastritis and stomach ulcers.

This invention also encompasses inflammation of columnar epithelialcaused or contributed to by surgery, allergy, chemical exposure, andphysical injury.

Methods of Screening for Anti-inflammatory Compounds

This invention provides a method of screening for a compound whichinhibits activation of inflammatory cells which interact with anepithelium. This method comprises pretreating the inflammatory cell withthe compound, placing the pretreated cell on one side of a preparedepithelial barrier having a chemotactic agent on the other side, anddetermining whether the compound inhibits the activation of theinflammatory cell. Nonlimiting examples of inflammatory cells areleukocytes such as polymorphonuclear leukocytes (PMN), eosinophils,T-lymphocytes, B-lymphocytes, natural killer cells, andmonocyte-macrophages. Inflammatory cell activation includes adhesion tothe epithelium, migration across the epithelium, release of bioactivemolecules, or a combination thereof.

The epithelial barrier can be constructed by growing epithelial cellsand forming a monolayer by controlling the growth media to preserve thepolarized phenotype. For example, T84 cells are grown as monolayers in a1:1 mixture of Dulbecco-Vogt modified Eagle's medium and Ham's F12medium supplemented with 15 mM Na⁺-HEPES buffer, pH 7.5, 1.2 g/l NaHCO₃,40 mg/l penicillin, 8 mg/l ampicillin, 90 mg/l streptomycin, and 5%newborn bovine serum.

Normal or inverted monolayers can be constructed using the commerciallyavailable insert system (Costar inserts, 0.33 cm², 5 μm polycarbonatefibers, Cambridge, Mass.). The larger pore size is crucial to allowinflammatory cells to penetrate the filter. Furthermore, the filter mustbe coated with Collagen I to allow epithelial cell attachment. Preparedmonolayers should be used within 6-14 days, since not only dophysiologic responses diminish with time, but also cell processes caneventually move through the 5 μm pores and result in a doubledmonolayer, with one monolayer on each side of the filter. The monolayermay be inverted or not, to allow screening for migration, adhesion, orrelease of bioactive molecules in both the apical-to-basal direction andthe basal-to-apical direction.

Nonlimiting examples of cells from which to form the epithelial barrierinclude: the intestinal cell lines Caco-2 (ATCC accession number HTB37), IEC-6 (ATCC accession number CRL 1592), T84 (ATCC accession numberCCL 248) or HT-29 (ATCC accession number HTB 38); the renal tubular celllines MDCK (ATCC accession numbers CCL 34 and CRL 6253) or LLC-PK₁ (ATCCaccession numbers CL 101 and CRL 1392); and isolated alveolar epithelialcells grown in primary culture.

The prepared epithelial barrier may optionally have a permeableartificial membrane on one side to prevent membrane-membrane contactbetween the epithelial barrier and the inflammatory cell. While thereare numerous artifical supports available, a preferable membrane made ofpolycarbonate may be obtained commercially from Costar Corp., Cambridge,Mass.

The epithelial barrier also may have cell-sized objects (approximately7-10 μm in diameter) located in the interstitial spaces between theepithelial barrier cells. These objects can be actual cells, or latexbeads. The latex beads can be inert or coated with one or more types ofactive molecules attached to the bead surface, such as marker molecules,signal molecules, or monoclonal antibodies. The inert beads areavailable commercially (Seradyne, Indianapolis, Ind.). The beads mimicthe physical presence of inflammatory cells. In addition, the coatedbeads provide a high local concentration of the coating molecule(s) andmimic the structural stability of cell-cell membrane contact.Furthermore, the beads provide a method of introducing bioactivemolecules of otherwise low solubility into the system for long periodsof time. The beads may be coated with a particular selected molecule,without undue experimentation, by methods known to those skilled in theart.

A chemotactic agent elicits the adhesion, migration, release of abioactive molecule, or combination thereof by the inflammatory cells onthe opposite side of the epithelial barrier. Nonlimiting examples of anappropriate chemotactic agent are: eicosanoids such as leukotriene B₄(LTB₄), 12S-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (12-HETE),and 5S-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid (5-HETE); IL-8,IGF-β, C5a, platelet activating factor (PAF), and N-formyl-Met-Leu-Phe(fMLP). In addition, any microbial pathogen-derived chemotactic factormay be used, since fMLP is a model attractant for bacterial chemotaxis.The amount of chemoattractant should be sufficient to elicit adhesion,migration, or release of a bioactive molecule in the absence of aninhibiting compound for the particular barrier system being used. Forexample, a concentration of 1 μM fMLP may be used.

Measuring the inhibition of inflammatory cell activation can be achievedin several ways. The relative number of migrating PMN cells can bemeasured, for example, by a myeloperoxidase assay. The effect of cellactivation, in the form of specific barrier dysfunction or abnormalelectrolyte transport, can also be evaluated with electrophysiologicalmeasurements of the electrical resistance of the epithelial barrier, theelectrical resistance of the epithelial cell membrane, and/or theendogenous cell current.

In a typical embodiment, the method would be a method of screening for acompound which modifies PMN adhesion to or migration across anintestinal epithelial barrier. This method comprises pretreating PMNwith the compound, placing the pretreated PMN on one side of theintestinal barrier having a chemotactic agent on the other side, anddetermining whether the compound modifies PMN adhesion to or migrationacross the barrier. The epithelial barrier may be modeled by columnarepithelial cells with features similar to those of natural cryptepithelial, such as but not limited to a monolayer of human intestinalepithelial cell line T84. The chemotactic agent is fMLP (1 μM). Thedetermination of the effectiveness of the compound is measured by therelative change in migration or adhesion of the PMN as measured by amyeloperoxidase assay. (Madara, J. L. et al. (1992) J. Tiss. Cult. Meth.14:209-216.) Experimental details of this embodiment of the screeningmethod are provided in Example 1 below.

This invention also provides a method of screening for a compound whichattentuates the effect of an activated inflammatory cell upon a columnarepithelium, thereby attenuating one or more deleterious perturbations.This method comprises: combining an inflammatory cell with a preparedepithelial barrier, pretreating this combination with the compound,adding an activating agent, and determining whether the deleteriousperturbations are attentuated by the. compound.

The activating agent is an agent which stimulates the activation of thethe inflammatory cell. Nonlimiting examples of an inflammatory cellactivating agent are: phorbol ester, a Ca⁺² ionophore,phytohemaglutinin, chemotactic agents as described above, and endotoxin.In addition, the activating agent may have an effect on both theinflammatory cell and the epithelial cell. Nonlimiting examples of thesekinds of activating agents are cytokines such as γ-IFN. The preparedepithelial barrier can be made as described above.

The attenuation can be measured in terms of electrical parameters suchas the electrical resistance of the epithelial barrier, the electricalresistance of the epithelial cell membrane, or the endogenous current,or combinations thereof. The relative attenuation is the comparison ofelectrical parameters in the presence and absence of the compound.

A typical embodiment of this method will be used to screen for acompound which reduces or eliminates the symptoms of secretory diarrheacaused by abnormal chloride secretion. The PMN-derived paracrine factorthat elicits chloride secretion from T84 intestinal epithelial cellmonolayers is 5′-adenosine monophosphate (5′-AMP). (Madara, J. L. et al.(1993) J. Clin. Invest. 91: 2320-2325.) The method. comprises: combiningan intestinal epithelial barrier with PMN cells, stimulating chloridesecretion by an intestinal epithelial cells with an amount of 5′-AMP oran agonist thereof; exposing the epithelial cells to the compound; anddetermining the attenuating effect of the compound upon the activationof the epithelial cells. The attenuation is measured by the electricalresistance of the epithelial barrier, the electrical resistance of theepithelial cell membrane, and/or the endogenous cell current.

Nonlimiting examples of 5′-AMP agonists are cyclic AMP, forskolin, andcarbachol. Nonlimiting examples of variable ranges appropriate for astandard dose-response curve are: 5′-AMP (10⁻⁸−10⁻³ M, in the apicaldirection; 10⁻⁷−10⁻² M in the basal direction); cAMP and forskolin(10⁻⁸−10⁻² M); and carbachol (10⁻⁸−10⁻³ M). For example, incrementalsteps of one-half log concentrations may be used. The amount of the5′-AMP or agonist should be an amount sufficient to elicit intestinalchloride secretion. The following Example 2 discloses the experimentaldetails for performing the electrophysiological measurements.

The intestinal epithelial barrier may be from, but is not limited to,any of the above mentioned intestinal cell lines, especially the T84cell line. In addition, the screened compound may be, for example, aneicosanoid such as a lipoxin or lipoxin analog. The lipoxin analog mayhave a longer tissue half-life than the corresponding lipoxin, or may beactively absorbed by the intestine, or both.

Lipoxins Lipoxin Analogs, and Combinations Thereof

Lipoxin compounds (e.g. natural lipoxins and lipoxin analogs) can beadministered to a subject for the treatment or prevention ofinflammation or inflammatory responses caused or contributed to byepithelial perturbations. Preferred lipoxin compounds are naturallipoxin A4 (LXA₄) and analogs thereof.

“Natural lipoxins” are lipoxygenase-derived, biologically activeeicosanoids produced by PMN, platelets, eosinophils and macrophages.(Samuelsson B., et al. (1987). Science 237: 1171-1176); (Dahlen S. E.,and C. N. Serhan (1991). In Lipoxygenases and Their Products, AcademicPress. New York, N.Y. 235-276). These compounds have been shown toelicit selective counterregulatory responses in human PMN in vitro,including the inhibition of leukotriene B₄ (LTB₄) and fMLP-stimulatedchemotactic responses across Boyden chambers (filters) (Lee T. H., etal. (1989). Clin Sci. 77: 195-203); (Lee T. H., et al. (1991). BiochemBiophys Res Comm 180: 1416-1421), blocking of Ca²⁺ mobilization(Springer T. A. (1990). Nature 346: 196-197), and inhibition ofLTD₄-induced adhesion to mesangial cells (Brady H. R., et al. (1990).Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F809-815). Invivo, lipoxins are potent inflammatory mediators which act to inhibitlymphocyte migration across vascular endothelia (Hedqvist P. J. et al.(1989). Acta. Physiol. Scand. 137: 571-572), decrease LTD₄-inducedvasoconstriction (Badr K. F., et al. (1989). Proc. Nat. Acad. Sci.U.S.A. 86: 3486-3442), and modulate LTD₄-induced airway obstruction,(Christie P. E., et al. (1992) Am Rev Respir Dis 145: 1281-1284).Lipoxins include the bioactive (5S, 14R,15S)-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid (LXB₄), andmore preferably, (5S, 6R,15S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (LXA₄).

In addition to natural lipoxins, lipoxin analogs are usefulantiinflammatory agents. “Lipoxin analogs” include compounds which arestructurally similar to natural lipoxins, compounds which share the samereceptor recognition site, compounds which share the same or similarlipoxin metabolic transformation region as lipoxin, and compounds whichare art-recognized as being analogs of lipoxin. Lipoxin analogs alsoinclude metabolites of lipoxin and lipoxin analogs. A nonlimitingexample of a lipoxin analog which inhibits PMN migration across anepithelial barrier is 11-trans-LXA₄. (See Example 1). Some lipoxinanalogs are sufficiently lipophilic to be actively absorbed by theintestine. Generally, lipophilic analogs will have relatively short(C₂-C₄) hydrocarbon groups occupying the C-16+position, as in thenatural lipoxin compound.

One particularly suitable class of lipoxin analogs for use in theinstant invention are those exhibiting a longer tissue half-life thancorresponding natural lipoxins. A “lipoxin analog having a longer tissuehalf-life than corresponding lipoxins” refers to a compound which has an“active region” that functions like the active region of a naturallipoxin (e.g. LXA₄ or LXB₄), but which has a “metabolic transformationregion” that differs from natural lipoxin. By“active region” is meantthe region of a natural lipoxin or lipoxin analog, which is associatedwith in vivo cellular interactions. The active region may bind the“recognition site” of a cellular lipoxin receptor or a macromolecule orcomplex of macromolecules, including an enzyme and its cofactor.Preferred lipoxin A₄ analogs have an active region comprising C5-C15 ofnatural lipoxin A₄. Preferred lipoxin B₄ analogs have an active regioncomprising C5-C14 of natural lipoxin B4.

The term “metabolic transformation region” refers to that portion of alipoxin, a lipoxin metabolite, or lipoxin analog including a lipoxinanalog metabolite, upon which an enzyme or an enzyme and its cofactorattempts to perform one or more metabolic transformations which thatenzyme or enzyme and cofactor normally transform on lipoxins. Themetabolic transformation region may or may not be susceptible to thetransformation. A nonlimiting example of a metabolic transformationregion of a lipoxin is a portion of LXA₄ that includes the C-13,14double bond or the C-15 hydroxyl group, or both.

The pathway of lipoxin metabolism includes dehydrogenation, reduction ofat least one unsaturated carbon-carbon bond, and/or ω-oxidation. Theseenzymatic transformation occur within the C-12 to C-20 portion of anLXA₄ analog, for example. Therefore, a lipoxin analog with a longertissue half-life may be designed with chemical modifications whichinhibit, resist, or raise the transition state energy of an analog orits metabolite for at least one of the metabolic transformations. Suchanalogs employ electronic effects at the relevant carbon atom, stericeffects, and/or potential suicide substrate moieties such as those thatallow covalent Michael addition to a metabolic enzyme.

Nonlimiting examples of a LXA₄ analog having a longer tissue half-lifethan LXA₄ include LXA₄ analogs with C-15 and/or C-16 substitutions suchas: mono- or di- hydroxyl, methyl, fluoromethyl, and fluoro; C-16substitutions such as phenyl, halo-substituted phenyl, and alkoxy; andC-19 or C-20 substitutions such as fluoromethyl, phenyl, and fluoro; and13-yne or 14-yne substitutions. It is known that the intestine activelyabsorbs lipophilic fatty acids, especially those two to four carbonatoms in length. (Binder, H. J. In, Gastrointestinal Disease, 4th ed.(Sleisenger, M. H., and Fordtran, J. S., eds.) W. B. Saunders Co.,Phildelphia, 1989, pp. 1022-1045.) In other embodiments, similarsubstitutions create structural analogs based on other lipoxins such asLXB₄.

In the most preferred embodiment of this invention, the compounds ofthis invetion have following structural formulas:

where R′ is H or CH₃.

In other preferred embodiments of this invention, the compounds of thisinvention have the following structural formulas:

This invention also contemplates use of combinations of lipoxins andlipoxin analogs. A nonlimiting example of a combination is a mixturecomprising a lipoxin analog x which inhibits one enzyme whichmetabolizes lipoxins and which optionally has specific activity with alipoxin receptor recognition site, and a second lipoxin analog y whichhas specific activity with a lipoxin receptor recognition site and whichoptionally inhibits or resists lipoxin metabolism. This combinationresults in a longer tissue half-life for at least y since x inhibits oneof the enzymes which metabolize lipoxins. Thus, the lipoxin actionmediated or antagonized by y is enhanced.

Methods of Making Lipoxins and Lipoxin Analogs

Lipoxins may be isolated as described (Serhan, C. N. et al. (1990)Methods in Enzymol. 187: 167) from biological sources, synthesized orobtained commercially. LXA₄ and LXB₄ are available from Biolmol, Inc.(Philadelphia, Pa.) and Cayman Biochemical (Ann Arbor, Mich.). LXA₄,LXB₄, and the 11-trans-LXA₄ isomer are available from CascadeBiochemical, Ltd (Berkshire, UK). Nonlimiting examples of the structuresand syntheses of both lipoxins and lipoxin analogs, including methylesters of lipoxin analogs, are illustrated in the following patents andpublications:

(1) Nicolaou, K. C. et al. Identification of a novel 7-cis-11-trans-LXA₄generated by human neutrophils: total synthesis, spasmogenic activitiesand comparison with other geometric isomers of lipoxins A₄ and B₄(1989). Biochim. Biophys. Acta 1003:44-53;

(2) Nicolaou, K. C. et al. Total synthesis of novel geometric isomers ofLXA₄ and LXB₄ (1989). J. Org. Chem. 54: 5527-5535;

(3) Nicolaou, K. C. et al. Lipoxins and related eicosanoids:biosynthesis, biological properties, and chemical synthesis (1991).Angew. Chem. Int. Ed. Engl. 30: 1100-1116;

(4) U.S. Pat. Nos. 4,576,758; 4,560,514; 5,079261; and 5,049,681; and

(5) JP Patent Nos. 3,227,922; 63,088,153; 62,198,677; and 1,228,994.

Preferred lipoxin analogues having a longer half-life than naturallipoxins can be prepared as described in the following Example 2

Methods of Treatment

This invention provides, in part, method of treating or preventinginflammation or an inflammatory response caused or contributed to by theactivation of inflammatory cells which interact with a columnarepithelium. The interaction between activated inflammatory cells and theepithelium results in one or more epithelial perturbations. Thisanti-inflammatory treatment is the administration to a subject of aneffective amount of a lipoxin, lipoxin analog, or combination thereof toinhibit the activation of the inflammatory cell such that the epithelialperturbation and inflammation or an inflammatory response aresignificantly reduced or eliminated.

A significant reduction of inflammation or an inflammatory responseincludes reducing or eliminating one or more of the symptoms associatedwith inflammation. For example, PMN transmigration stimulateselectrogenic chloride secretion, which is the basis of secretorydiarrhea, one of the symptoms of inflammatory bowel diseases. (Nash, S.et al. (1991). J.Clin. Invest. 87: 1474-1477.) Additional nonlimitingexamples of symptoms of inflammatory bowel diseases are crampingabdominal pain, malabsorption, dehydration, bloody stool, or fever. Inaddition to the inflammatory bowel disease listed above, bowelinflammation may also result from surgery, allergy, chemical exposure,or physical injury. Reduction of epithelial perturbation can alsoinclude inhibition of inflammatory cell activation. For example, areduced perturbation can be the inhibition of PMN migration in thebasal-to-apical direction represented by a decrease of at least about25%.

Lipoxins include LXA₄ or LXB₄. The lipoxin analog can have a longertissue half-life than the corresponding natural lipoxin. The lipoxinanalog can also be lipophilic. The lipoxin analog can also be activelyabsorbed by the intestine. Lipoxins, lipoxin analogs, and combinationsof lipoxins as used in these methods of treatment are defined above inthe preceding two sections.

This invention also provides a method for the treatment or prevention ofone or more of the symptoms of inflammatory diseases of columnarepithelia. In this method, the epithelial perturbations which cause orcontribute to these symptoms may or may not be mediated by inflammatorycells. This method of treatment comprises the administration to asubject of an effective amount of a lipoxin, lipoxin analog, orcombination thereof such that the epithelial inflammation orinflammatory response is significantly reduced or eliminated.

A significant reduction of inflammation or an inflammatory responseincludes reducing or eliminating one or more of the symptoms associatedwith inflammation. For example, abnormal chloride secretion causes orcontributes to secretory diarrhea, a symptom of inflammatory boweldiseases. 5′AMP elicits chloride secretion from T84 intestinalepithelial cell monolayers, in a manner which may not always bedependent upon PMN. (Madara, J. L. et al. (1993) J.Clin. Invest.91:2320-2325.) Additional nonlimiting examples of symptoms ofinflammatory bowel diseases are cramping abdominal pain, malabsorption,dehydration, bloody stool, or fever.

Lipoxins include LXA₄ or LXB₄. The lipoxin analog may havecharacteristics such as a longer tissue half-life than the correspondingnatural lipoxin, be lipophilic, or be actively absorbed by theintestine, or a combination thereof Lipoxins, lipoxin analogs, andcombinations of lipoxins as used in these methods of treatment aredefined above.

In one embodiment, the lipoxin or lipoxin analog independently acts tomodulate epithelial perturbations, such as chloride ion secretion.Without intending to be bound, it is speculated that lipoxins andlipoxin analogs, independent of PMN activation, can decrease chlorideion secretion to an extent that secretory diarrhea is significantlyreduced.

Pharmaceutical Compositions and Packaged Drugs

This invention also encompasses pharmaceutical compositions and packageddrugs containing lipoxins, lipoxin analogs, salts thereof, andcombinations thereof for the treatment of inflammation and inflammatoryresponses in a subject. In one embodiment of this invention, thepharmaceutical compositions and packaged drugs are for the treatment orprevention of the columnar epithelial perturbations related to PMNactivation in inflammatory bowel diseases.

The term “subject” is intended to include living organisms susceptibleto conditions or diseases caused or contributed to by inflammation andinflammatory responses. Examples of subjects include humans, dogs, cats,cows, goats, and mice. The term “subject” is further intended to includetransgenic species.

The term “pharmaceutically acceptable salt” is intended to includeart-recognized pharmaceutically acceptable salts. These non-toxic saltsare usually hydrolyzed under physiological conditions, and includeorganic and inorganic bases. Examples of salts include sodium,potassium, calcium, ammonium, copper, and aluminum as well as primary,secondary, and tertiary amines, basic ion exchange resins, purines,piperazine, and the like. The term is further intended to include estersof lower hydrocarbon groups, such as methyl, ethyl, and propyl. In thisparagraph, the next paragraph, and in the discussion of methods oftreatment and pharmaceutical compositions, it should be understood thatreferences to lipoxin analogs are meant to include correspondingpharmaceutically acceptable salts.

The term “pharmaceutical composition” comprises one or more naturallipoxin or lipoxin analog as an active ingredient(s), or apharmaceutically acceptable salt(s) thereof, and may also contain apharmaceutically acceptable carrier and optionally other therapeuticingredients. The compositions include compositions suitable for oral,rectal, ophthalmic, pulmonary, nasal, dermal, topical, parenteral(including subcutaneous, intramuscular and intravenous) or inhalationmodes of administration. The most suitable route in any particular casewill depend on the nature and severity of the conditions being treatedand the nature of the active ingredient(s). The compositions may bepresented in unit dosage form and prepared by any of the well-knownmethods.

Appropriate dosage regimes for treating a particular disease orcondition associated with columnar epithelial inflammation can bedetermined empirically by one of skill in the art and may be adjustedfor the purpose of improving the therapeutic response. For example,several divided dosages may be administered daily or the dose may beproportionally reduced over time. A person skilled in the art normallymay determine the effective dosage amount and the appropriate regime. Aless potent lipoxin analog composition may be selected to treat mild orhighly localized inflammation, while a larger dosage or more potentlipoxin analog may be selected to treat severe or widespreadinflammatory episodes. An “effective anti-inflammatory amount” of alipoxin containing pharmaceutical composition for treating a disease orcondition associated with a columnar epithelial inflammation shall meanthat amount that ameliorates the inflammation and eliminates the syptomsof the disease. An “effective anti-diuretic amount” of a lipoxincontaining pharmaceutical composition is that amount that restorestransportation of fluid, electrolytes, or nutrients by a columnarepithelium to the normal, homeostatic level.

The term “packaged-drug” is meant to include one or more dosages of aneffective pharmaceutical composition of a lipoxin, a lipoxin analog,salt thereof or combination thereof, a container holding the dosage(s),and instructions for administering the dosage(s) to a subject fortreatment or prevention of inflammation or an inflammatory response.

The present invention is further illustrated by the following examplewhich should in no way be construed as being further limiting. Thecontents of all references and issued patents cited throughout allportions of this application including the background are expresslyincorporated by reference.

EXAMPLE 1 Lipoxin A₄ Modulates Migration of Human PMNs Across Intestinal

Epithelial Monolayers.

Lipoxins Synthetic LXA₄, LXB₄, and 11-trans-LXA₄ were obtained fromCascade Biochem Ltd. (Berkshire, United Kingdom). Concentrations weredetermined from extinction coefficients as described in Sheppard K-A.,et al. (1992). Biochimica et Biophysica Acta 1133: 223-234. Alleicosanoid stock solutions were stored at −70° C. in methanol (AmericanScientific Products). Eicosanoids were diluted in modified HBSS to aconcentration of 1 μM prior to all experiments. PMN or T84 monolayerswere exposed to lipoxins at indicated concentrations and allowed toincubate at 37° C. for the indicated period of time. Vehicle controlsconsisted of dilutions of the solvent (ethanol) equivalent to thehighest concentration of lipoxin used in any given experiment (0.01%).

Cell culture Approximately 350 epithelial monolayers were used for thesestudies. T84 intestinal epithelial cells (passages 70-95) were grown andmaintained as confluent monolayers on collagen coated permeable support.Monolayers were grown on 0.33 cm² ring-supported polycarbonate filters(Costar Corp., Cambridge, Mass.) and utilized 6-14 days after plating asdescribed in Example 2. Transepithelial resistance to passive ion flowwas measured as described in Parkos C. A., et al. (1991). J. Clin.Invest. 88: 1605-1612); and Parkos C. A., et al. (1992). J. Cell. Biol.117: 757-764). Inverted monolayers used to study migration of PMN in thebasolateral-to-apical direction were constructed as described in ParkosC. A., et al. (1991). J. Clin. Invest. 88: 1605-1612).

Migration assay: The PMN transepithelial migration assay has beendetailed in Nash S., et al. (1987). J. Clin. Invest. 80: 1104-1113; NashS., et al. (1991). J. Clin. Invest. 87: 1474-1477); Parkos C. A., et al.(1991). J. Clin. Invest. 88: 1605-1612); and Parkos C. A., et al.(1992). J. Cell. Biol. 117: 757-764. Briefly, human PMN were isolatedfrom normal human volunteers and suspended in modified HBSS (withoutCa²⁺ and Mg²⁺, with 10 mM Hepes, pH 7.4, Sigma) at a concentration of5×10⁷/ml. Prior to addition of PMN, T₈₄ monolayers were extensivelyrinsed in HBSS to remove residual serum components. Migration assayswere performed by the addition of PMN (40 μl) to HBSS (160 μl) in theupper chambers after chemoattractant (1 μM fMLP in HBSS) was added tothe opposing (lower) chambers. Unless otherwise indicated, PMN were notwashed free of LXA₄ prior to addition to monolayers, and therefore, afive-fold dilution of lipoxin was present during the migration assay.For apical-to-basolateral migration experiments, PMN (2×10⁶) were addedat time 0. Migration in the basolateral-to-apical direction, whilequalitatively similar, is substantially more efficient than in theapical-to-basolateral direction. (Parkos C. A., et al. (1991). J. Clin.Invest. 88: 1605-1612). Therefore, 5-fold fewer PMN (4×10⁵) were addedwhen migration proceeded in the basolateral-to-apical direction in orderthat baseline migration signals be approximately equivalent in bothdirections. (Parkos C. A., et al. (1991). J. Clin. Invest. 88:1605-1612). Migration was allowed to proceed for 120 minutes, unlessotherwise noted. All PMN transepithelial migration experiments wereperformed in a 37° C. room to ensure that epithelial monolayers,solutions, plasticware, etc., were maintained at uniform 37° C.temperature.

When used, inhibitors to cyclooxygenase (indomethacin, Sigma),leukotriene biosynthesis (MK886, a kind gift from Merck Frosst),G-proteins (pertussis toxin, Calbiochem), or protein kinase C (H7,Sigma; staurosporine, Sigma) were pre-incubated with PMN at indicatedconcentrations for 15 min at 37° C. Inhibitors were washed free from PMNby two washes with HBSS. PMN were subsequently exposured to LXA₄ (10 nM)and PMN transepithelial migration was assessed as described above in theapical-to-basolateral direction.

Migration was quantitated by assaying for the PMN azurophilic granulemarker myeloperoxidase (MPO) as described previously (Parkos C. A., etal. (1991). J. Clin. Invest. 88: 1605-1612). Following each migrationassay, non-adherent PMN were extensively washed from the surface of themonolayer and PMN cell equivalents (PMN CE), estimated from a standardcurve, were assessed as the number of PMN's associated with themonolayer, the number which had completely traversed the monolayer (ie.across the monolayer into the reservoir bath), as well as the totalnumber of trnsmigrating PMN (the sum of monolayer andreservoir-associated PMN).

Data Presentation: Individual experiments were performed using largenumbers of uniform groups of monolayers and PMN from individual blooddonors on individual days. PMN isolation was restricted to fivedifferent blood donors (repetitive donations) over the course of thesestudies. Myeloperoxidase assay data were compared by two-factor analysisof variance (ANOVA) or by comparison of means using Student's t-Test.PMN migration results are represented as PMN CE derived from a dailystandard PMN dilution curve. Monolayer-associated PMN are represented asthe number of PMN CE per monolayer and reservoir-associated PMN (ie. PMNwhich had completely traversed the monolayer into the lower chamber) arerepresented as the number of PMN CE/ml (total volume of 1 ml). Valuesare expressed as the mean and s.e.m. of n experiments.

RESULTS LXA₄ Exposure to T84 Epithelial Monolavers does not AlterSubsequent fMLP-induced PMN Migration

PMN can be induced to transmigrate across T₈₄ epithelial monolayers inresponse to a transepithelial gradient of the chemotactic peptide fMLP(1 μM).(Nash S., et al. (1987). J. Clin. Invest. 80:1104-1113); (ParkosC. A., et al. (1991). J. Clin. Invest. 88:1605-1612). To determinewhether LXA₄ exposure to T₈₄ intestinal epithelial cells influencedsubsequent PMN migration, epithelial cell monolayers were incubated withLXA₄ at a concentration 10 nM for 15 min at 37° C. (conditions whichelicit significant effects when PMN's are pre-exposed to LXA₄, seebelow), with and without removal of LXA₄ from monolayers, followed byaddition of untreated PMN's under chemotactic conditions. In theseexperiments, PMN migration across T84 monolayers exposed to LXA₄ did notdiffer from vehicle control (14.8±1.4 vs 15.7±1.8×10⁴ PMN CE/ml forcontrol and LXA₄ exposed monolayers, n=6 each, n.s.). Removal of LXA₄from monolayers by washing thrice with HBSS prior to addition of PMN hadno apparent effect on the total number of transmigrating PMN(14.3±1.1×10⁴ PMN CE/ml, n=6, n.s. compared to either control or LXA₄exposed monolayers).

In addition, exposure of intestinal epithelial cells to LXA₄ did notsignificantly influence the integrity of T84 epithelial monolayers. Toexamine this, transepithelial resistance to passive ion flow wasassessed prior to, and after addition of 10 nM LXA₄ to T84 intestinalepithelial monolayers for 2 hrs (simulated conditions for entiremigration assay period). During this period, transepithelial resistancedid not significantly decrease following addition of LXA₄ (baselineresistance 1255±56 ohm-cm² and 1089±108 ohm-cm² after 2 hr, n=8, n.s.).These results suggest that monolayer integrity, as assessed bytransepithelial resistance, was not affected by LXA₄ treatment and thatepithelial pre-exposure to LXA₄ has no subsequent effect on fMLP-inducedPMN migration.

LXA₄ does not Stimulate Migration

To investigate whether LXA₄ could serve to stimulate PMN migration inthis assay system, dilutions of LXA₄ in the range of 0.01-10 nM wereplaced in the lower chamber of migration wells. Untreated PMN's wereadded to the upper chamber and assessed for chemotactic capacity towardLXA₄ in the apical-to-basolateral direction. LXA₄ was no more effectivethan HBSS in promoting PMN migration; compared to fMLP (1 μM), PMNmigration toward LXA₄ resulted in a total of 6±2.1, 9±3.6, 6±2.4, and12±1.4% of fMLP-induced PMN migration for 0.01, 0.1, 1.0, and 10 nMLXA₄, respectively. In the absence of a chemotactic gradient (HBSS),10±2.9% of fMLP-induced migration occurred. These results indicate thatLXA₄, in the concentrations tested, did not stimulate PMNtransepithelial migration.

Pre-exposure of PMN to LXA₄ Enhances fMLP-induced PMN Migration in theApical-to-basolateral Direction

To determine if PMN exposure to LXA₄ alters subsequent fMLP-inducedmigration, PMN were incubated 10 nM LXA₄ for 15 minutes, then addeddirectly to the apical surface of T84 epithelial monolayers, andsubsequently assessed for their ability to traverse T84 epithelialmonolayers using a myeloperoxidase assay. (Parkos C. A., et al. (1991).J. Clin. Invest. 88: 1605-1612). PMN (5×10⁷/ml) were pre-incubated with10 nM LXA₄ for 15 min. at 37° C. and layered on the apical surface ofwashed T84 epithelial monolayers at a density of 2×10⁶/monolayer. PMNwere driven to transmigrate basolaterally under the influence of a 1 μMgradient of fMLP.

Results were obtained by harvesting the PMN specific enzymemyeloperoxidase (MPO) from washed monolayers, lower reservoirs and totalMPO activity after 120 min, relative to a known standard number of PMN.Since tight junctions are the rate limiting barrier to passiveparacellular permeation, transmigration is defined as movement of PMNacross the tight junction. Since monolayer-associated PMN were largelybelow the tight junction (see results), total transmigration in theapical-to-basolateral direction equals the sum of PMN in the oppositereservoir plus monolayer PMN. Data are pooled from 9 individualmonolayers in each condition and results are expressed as the mean andSEM.

PMN pre-exposure to LXA₄ resulted in significantly increased PMNtransepithelial migration in the apical-to-basolateral direction.Increased PMN migration was evident in both monolayer-associated PMNnumbers (2.98±0.57 vs. 6.93±1.77×10⁴ PMN CE/monolayer for vehiclecontrol and LXA₄ exposed PMN, respectively, p<0.001), as well as thenumber of PMN which completely traversed the epithelial monolayer(6.61±0.50 vs. 11.02±2.91×10⁴ PMN CE/ml for vehicle control and LXA₄exposed, respectively, p<0.1), resulting in a nearly 2-fold increase inthe total number of transmigrating PMN (9.58±1.05×10⁴ PMN CE/ml forvehicle control and 17.95±2.15×10⁴ PMN CE/ml for LXA₄ pre-exposed PMN,p<0.01). As reported previously, (Parkos C. A., et al. (1991). J. Clin.Invest. 88: 1605-1612), examination of 1 μm T84 epithelial monolayersections have revealed that PMN are only rarely associated with theapical epithelial surface and the majority of monolayer associated PMNare found subjunctionally, indicative of migration. Therefore,monolayer-associated PMN in this apical-to-basolateral assay areconsidered transmigrated across the tight junction, the rate limitingbarrier in PMN transepithelial migration. (Parkos C. A., et al. (1991).J. Clin. Invest. 88: 1605-1612).

To further characterize this transmigratory event, PMN were pre-exposedto LXA₄ (10 nM) for various periods of time and subsequently assessedfor their ability to transmigrate across T84 epithelial monolayers inthe apical-to-basolateral direction. PMN (5×10⁷/ml) were pre-incubatedwith 10 nM LXA₄ for various periods of time in the range of 0-60 min at37° C. or pre-incubated with various indicated concentrations of LXA₄for 15 min. at 37° C. and layered on the apical surface of washed T84epithelial monolayers at a density of 2×10⁶/monolayer. PMN were drivento transmigrate basolaterally under the influence of a 1 μM gradient offMLP. Results were again obtained by harvesting the PMN specific enzymemyeloperoxidase (MPO) from washed monolayers, lower reservoirs and totalMPO activity after 120 min, relative to a known standard number of PMN.Data are pooled from 7-10 individual monolayers in each condition andresults are expressed as the mean and SEM.

Pre-exposure of PMN to LXA₄ resulted in increased total PMN migrationafter a LXA₄ pre-exposure period of 5-30 min. (compared to vehiclecontrols, for PMN's pre-exposed to 10 nM LXA₄, migration increased by50, 68 and 51% at 5, 15 and 30 min. exposure times, respectively, allp<0.025). Migration had returned to vehicle control values by 45 and 60minutes of PMN pre-exposure to LXA₄. Pre-exposure of PMN to LXA₄ wasfound to be a necessary prerequisite for LXA₄ action on stimulating PMNmigration. Indeed, exposure of PMN's to 10 nM LXA₄ immediately prior totheir addition to epithelial monolayers (ie. 0 min. pre-exposure)resulted in no effect on subsequent fMLP-induced PMN migration(16.34±4.07 vs. 16.61±3.56×10⁴ total PMN CE/ml for vehicle control andLXA₄ pre-exposure for 0 min., respectively, n.s.). The LXA₄ pre-exposuretime-dependent enhancement of subsequent neutrophil migration waslargely due to reservoir-associated PMN (11.06±3.05 vs 18.02±3.35,19.96±3.18, 19.64±3.54×10⁴ PMN CE/ml for vehicle control and PMN LXA₄pre-exposure times of 5, 15 and 30 minutes, respectively, two-factorANOVA p<0.01). However, a significant increase in the number ofmonolayer-associated PMN occurred at 15 minutes of LXA₄ pre-exposure(4.17±1.17 for vehicle control vs 7.55±0.71×10⁴ PMN CE/monolayer forPMN's exposed to 10 nM LXA₄, p<0.01).

The effect of LXA₄ pre-exposure to PMN and subsequent PMNtransepithelial migration in the apical-to-basolateral direction wasfound to be concentration dependent. Pre-exposure of PMN to LXA₄concentrations in the range of 1.0 pM-10 nM for 15 minutes at 37° C.elicited increased PMN migration at doses of 0.1, 1.0 and 10 nM finalconcentrations. Similar to the time-course data presented above,LXA₄-elicited stimulation of PMN migration was manifest as an increasein the number of PMN in lower reservoirs (11.06±3.05 vs 20.38±4.83,19.96±4.83, 15.43±4.65, 14.11±3.01, and 13.71±4.14×10⁴ PMN CE/ml forvehicle control and PMN LXA₄ pre-exposure doses of 10, 1, 0.1, 0.01, and0.001 nM, respectively, for 15 min., 37° C., two-factor ANOVA, p<0.025).

To determine whether the stimulatory action of LXA₄ was presentthroughout the incubation period, PMN's were pre-exposed to LXA₄ (10 nM,15 min), layered on the apical surface of T84 monolayers and driven totransmigrate basolaterally. Monolayers were harvested at various timepoints during migration and assayed for PMN by myeloperoxidase content.PMN (5×10⁷/ml) were pre-incubated with 10 nM LXA₄ for 15 min. at 37° C.and layered on the apical surface of washed T84 epithelial monolayers ata density of 2×10⁶/monolayer. PMN were driven to transmigratebasolaterally under the influence of a 1 μM gradient of fMLP.

Results were obtained by assaying the PMN specific enzymemyeloperoxidase (MPO), relative to a known standard number of PMN. TotalMPO activity (including reservoir- and monolayer-associated MPOactivity) was tested. Data were pooled from 6 individual monolayers ineach condition and results were expressed as the mean and SEM.

The stimulatory effect of LXA₄ on PMN migration in theapical-to-basolateral direction was present by 45 min after addition ofPMN (0.06±0.04 vs 1.51±0.12×10⁴ PMN CE/ml for vehicle control and PMNexposed to LXA₄, respectively, p<0.05), and was maintained throughoutthe 135 min experimental period (two-factor ANOVA, p<0.01).

To assess whether pre-exposure of PMN to LXA₄ was reversible, PMN wereincubated with 10 nM LXA₄ for 15 minutes, washed twice in Ca²⁺- andMg²⁺-free HBSS, and assessed for their ability to migrate acrossmonolayers of T84 epithelial cells. in the apical-to-basolateraldirection. Here, PMN exposed to LXA₄ did not differ from control intheir ability to migrate across T84 epithelial monolayers (14.1±0.4 vs14.5±0.3×10⁴ PMN CE/ml for control and LXA₄ exposure followed bywashout, respectively, n=6 each, n.s.). In the presence of LXA₄, a totalof 18.6±1.0×10⁴ PMN CE/ml migrated (n=6, p<0.025 compared to control andwashout control), suggesting that LXA₄-induced enhancement of PMNmigration in the apical-to-basolateral direction requires the presenceof LXA₄.

A final determination was whether PMN-conditioned LXA₄ andepithelial-conditioned LXA₄ maintained its ability to enhance PMNmigration in the apical-to-basolateral direction. Samples of LXA₄ (10nM) were incubated with either PMN or T84 epithelial cells for 15 or 45minutes. Supernatants were harvested and subsequently exposed to PMN for15 min and added to the apical surface of T84 monolayers undertransmigratory conditions (1 μM fMLP transepithelial gradient) for 2 hrsat 37° C. Compared to PMN pre-exposed to HBSS (11.3±2.2×10⁴ total PMNCE/ml), PMN pre-exposed to PMN-conditioned LXA₄ (15 min) resulted in atotal PMN migration 18.0±2.1×10⁴ PMN CE/ml (n=3, p<0.05 compared tocontrol). PMN pre-exposed to epithelial-conditioned LXA₄ (15 min)resulted in a total PMN migration 16.8±3.3×10⁴ PMN CE/ml (n=3, p<0.05compared to control). Supernatants from PMN-conditioned LXA₄ (45 min)were not effective in enhancing PMN migration in theapical-to-bagolateral direction (12.36±3.1×10⁴ total PMN CE/ml comparedto HBSS control of 14.3±3.2×10⁴ total PMN CE/ml, n=3, p=n.s.). Theseresults suggest that enhancement of PMN migration in theapical-to-basolateral direction involves a step which is subsequent toPMN pre-incubation with LXA₄.

PMN pre-exposure to LXA₄ Decreases fMLP-induced PMN Migration in theBasolateral-to-apical Direction

Quantitative as well as qualitative differences can exist in PMNtransepithelial migration depending on the direction of migration. Toinvestigate the effect of LXA₄ on the polarity of migration, invertedmonolayers (which permit basolateral-to-apically directed migration)were prepared.

PMN (1×10⁷/ml) were pre-incubated with 10 nM LXA₄ for 15 min. at 37° C.and layered on the basolateral surface of washed T84 epithelialmonolayers (i.e. inverted monolayers) at a density of 4×10⁵/monolayer.PMN were driven to transmigrate apically under the influence of a 1 μMgradient of fMLP.

Results were obtained by harvesting the PMN specific enzymemyeloperoxidase (MPO) from lower reservoirs and washed monolayers after120 min, relative to a known standard number of PMN. Since tightjunctions are the rate limiting barrier to passive paracellularpermeation, transmigration is defined as movement of PMN across thetight junction. Since monolayer-associated PMN were largely below thetight junction, total transmigration in the basolateral-to-apicaldirection equates with PMN in the opposite reservoir only. Data werepooled from 9 individual monolayers in each condition and results wereexpressed as the mean and SEM.

Pre-exposure of PMN to LXA₄ (10 nM) for 15 minutes markedly decreasedPMN migration in the basolateral-to-apical direction. Unlike the resultsfound in the apical-to-basolateral direction, migration of PMNs in thisphysiologically relevant direction was significantly decreased comparedto vehicle controls (28.02±3.08 vs 18.77±1.48×10⁴ PMN/ml for control andPMN pre-exposed to 10 nM LXA₄ for 15 min, respectively, p<0.01).Migration in the basolateral-to-apical direction resulted in nosignificant difference in the number of monolayer-associated PMN'sfollowing pre-exposure to LXA₄ (2.01±0.20 vs 2.00±0.31 for control andPMN exposed to LXA₄, respectively, p=n.s.). This polarized action ofLXA₄ was confirmed by performing parallel apical-to-basolateral andbasolateral-to-apical migration experiments using T84 cells from thesame plating and same passage and using PMN from the same donors onthree separate occasions.

A time course of LXA₄ pre-exposure to PMN was next performed forbasolateral-to-apical directed migration. PMN (1×10⁷/ml) werepre-incubated with various indicated concentrations of LXA₄ for 15 min.at 37° C. and layered on the basolateral surface of washed T84epithelial monolayers at a density of 4×10⁵/monolayer. PMN were drivento transmigrate basolaterally under the influence of a 1 μM gradient ofFMLP.

Results were obtained by harvesting the PMN specific enzymemyeloperoxidase (MPO) from lower reservoirs and washed monolayers after120 min, relative to a known standard number of PMN. Data were pooledfrom 9-12 individual monolayers in each condition and results areexpressed as the mean and SEM.

Similar to the apical-to-basolateral direction, decreasedtransepithelial migration was present at 15 minutes of PMN pre-exposureto 10 nM LXA₄ (11.07±1.83 for control vs. 6.29 1.21×10⁴ PMN/ml, p<0.01).No differences in the number of monolayer-associated PMN were present atany period of LXA₄ exposure. Dose-response experiments (all 15 min.pre-exposure) revealed that pre-exposure of PMN to 10 and 1 nM LXA₄resulted in a significantly reduced number of transmigrating PMN in thebasolateral-to-apical direction (11.07±1.83×10⁴ PMN/ml for controlsamples vs 6.29±1.21 and 6.99±1.33×10⁴ PMN/ml following pre-exposure to10 and 1 nM LXA₄, respectively, both p<0.025). Again, this diminishedtransmigratory response in the basolateral-to-apical direction wasassociated with reservoir-associated PMN only, with no apparent effecton the number of monolayer-associated PMN.

It was also determined whether PMN-conditioned LXA₄ orepithelial-conditioned LXA₄ were effective in decreasing PMN migrationin the basolateral-to-apical direction. Similar to the results in theapical-to-basolateral direction (see above), PMN pre-exposed to eitherPMN-conditioned LXA₄ (8.07±1.63 vs. buffer control 13.18±1.91×10⁴PMN/ml, n=4, p<0.025) or epithelial-conditioned LXA₄ (9.01±1.76 comparedto buffer control of 14.23±2.06×10⁴ PMN/ml, n=4, p<0.04) maintainedactivity which decreased PMN transepithelial migration in thebasolateral-to-apical direction.

Pre-exposure of PMN to structurally related lipoxins

To investigate the specificity of LXA₄ causing decreased migration inthe physiological direction, the effects of PMN exposure to LXB₄ and11-trans-LXA₄ were also examined. PMN (1×10⁷/ml) were pre-incubated with10 nM LXA₄, LXB₄ or 11-trans-LXA₄ for 15 min. at 37° C. and layered onthe basolateral surface of washed T84 epithelial monolayers (i.e.inverted monolayers) at a density of 4×10⁵/monolayer. PMN were driven totransmigrate apically under the influence of a 1 μM gradient of fMLP.Results were obtained by harvesting the PMN specific enzymemyeloperoxidase (MPO) after 120 min, relative to a known standard numberof PMN. Total MPO activity (including reservoir- andmonolayer-associated MPO activity) was expressed as the percent PMNmigration inhibition. Data were pooled from 7 individual monolayers ineach condition and results were expressed as the mean and SEM.Pre-exposure of PMN's to 10 nM LXB₄, 11-trans-LXA₄ produced a 7±4%(p=n.s. compared to vehicle control) and 16±6% (p<0.05) inhibition ofPMN migration, respectively, while LXA₄ inhibited migration by 28±4%(p<0.01). These observations suggest structural specificity for LXA₄.

Effect of Inhibitors on LXA₄-elicited Enhancement of PMN TransepithelialMigration in the Apical-to-basolateral Direction

To determine whether LXA₄-induced modulation of PMN migration could bepharmocologically altered, a series of experiments were done in whichPMN were exposed to specific inhibitors, washed free of inhibitor andsubsequently assayed for the LXA₄ effect on PMN transepithelialmigration in the apical-to-basolateral direction.

Pre-exposure of PMN to indomethacin (50 μM, 15 min, 37° C.), acyclooxygenase inhibitor, (Smolen J. E., and G. Weissman (1980).Biochem. Pharmacol. 29: 533-538), did not effect baseline PMN migrationin the presence of a transepithelial gradient of fMLP (109±13% vehiclecontrol, n=6, p=n.s. compared to untreated control). Likewise,pre-exposure of PMN to indomethacin followed by exposure to LXA₄ (10 nM)did not alter the LXA₄-elicted increase in fMLP-driven PMN migration inthe apical-to-basolateral direction (61±11% increase vs 54±7% increaseover control for LXA₄ treated PMN with and without indomethacin,respectively, p=n.s.). Likewise, PMN pre-treatment with the compoundMK886 (10 ng/ml), a specific inhibitor of leukotriene generation,(Gillard J., et al. (1989). Can. J. Physiol. Pharmacol. 67: 456-464),did not alter baseline fMLP-driven PMN migration and did not effect theLXA₄-elicited increase in PMN transepithelial migration.

Staurosporine, a potent inhibitor of protein kinase C (PKC), (Sako T.,et al. (1988). Cancer Res. 48: 4646-4650), was assessed for its abilityto inhibit the LXA₄ effect. Interestingly, staurosporine alone (10 nMfinal concentration) was found to inhibit PMN transepithelial migration(93±5% inhibiton vs.vehicle control, n=3, p<0.001). These data were alsoconfirmed using the PKC inhibitor H7 (Nakadate, T., et al. (1989). Mol.Pharmacol. 36: 917-924.) (100 μM final concentration, 91±4% inhibitioncompared to vehicle control, n=3, p<0.001). Likewise, the LXA₄-elicited(10 nM) increment in migration was sensitve to staurosporine (89±7%inhibition compared to vehicle control, n=3, p<0.001). Pre-exposure ofPMN to pertussis toxin, Nigam, S., et al. (1990). J. Cell Physiol 143:512-523), (2μg/ml) also inhibited baseline fMLP driven migration (91±8%inhibition compared to vehicle control, n=6, p<0.001). The LXA₄-elicited(10 nM) increase in PMN migration (46±3% increase compared to control,p<0.01) was also sensitive to PMN pre-exposure to pertussis toxin (87±4%inhibition of control, n=6, p<0.001).

Next assessed was the possibility of differential sensitivity tostaurosporine for baseline and LXA₄-stimulated increases in fMLP-drivenPMN migration. Staurosporine inhibited baseline PMN transepithelialmigration in a dose-dependent manner (94±4%, 96±9%, 67±11%, 54±8%,35±11% and 11±6% inhibition compared to vehicle controls forconcentrations of 100, 10, 1, 0.1, 0.01 and 0.001 nM staurosporine,respectively, p<0.01 by ANOVA). From this dose response, a concentrationwas selected which was approximately half-maximal in inhibiting PMNmigration (0.1 nM, see above). PMN were then pre-exposed tostaurosporine (0.1 nM, 15 min, 37° C.), washed free of inhibitor, andsubsequently assessed for the LXA₄ effect on PMN transepithelialmigration. Here, the LXA₄-elicited increase in transepithelial migrationof PMN was observed to be sensitive to PKC inhibition, since therelative inhibition by staurosporine was equivalent with and withoutLXA₄ (54±6% and 49±7% decrease in total PMN migration for staurosporinetreated PMN in the presence and absence of LXA₄, respectively, p=n.s.;both decreased compared staurosporine untreated controls, n=6, p<0.025).

These results indicate that LXA₄-elicited increases in PMN migration inthe apical-to-basolateral direction are not sensitive to inhibition ofthe cyclooxygenase pathway or the specific inhibition leukotrienegeneration, but is sensitive to inhibitors of PKC.

DISCUSSION

During inflammatory processes, PMN are recruited from the blood bysignals derived at inflammatory sites. At sites of acute inflammation,PMN finction may be regulated by a variety of inflammatory signals,including both protein- and lipid-derived signals. PMN function atorgan-specific sites, including the intestine, are thought to contributeto epithelial dysfunction during disease. Here it is reported for thefirst time that the arachidonic acid-derived eicosanoid, LXA₄, modulatesPMN migration across a model human intestinal epithelium. In addition,it is here reported that LXA₄ exerts an effect on migration in apolarized fashion.

LXA₄ enhances PMN transepithelial migration in the apical-to-basolateraldirection. For technical reasons, previous studies of PMNtransepithelial migration have focused on “non-physiologically” orientedmonolayers, in which leukocyte migration is in the apical-to-basolateraldirection, (Nash S., et al. (1987). J. Clin. Invest. 80: 1104-1113),(Migliorisi G. E., et al. (1988). J. Leukocyte Biol 44: 485-492),(Parkos C. A., et al. (1991). J. Clin. Invest. 88: 1605-1612), (Evans C.W., et al. (1983) Br. J. Exp. Pathol. 64: 644-654). PMN pre-exposed toLXA₄ and driven to transmigrate across epithelial monolayers orientednon-physiologically resulted in enhanced PMN migration.

The action of LXA₄ was found to be specific for PMN, and not epithelialcells, since enhanced PMN migration in this direction was dose- andtime-dependent, and no measureable effects on PMN transepithelialmigration were observed when epithelial monolayers were pre-exposed toLXA₄ under conditions which promoted enhanced PMN migration. Theseresults are consistent with previous studies which report that LXA₄, insimilar concentrations used here, was capable of activating PMN invitro. In this model system, LXA₄ enhanced PMN migration in a mannerindependent to that of fMLP, since in all conditions PMN migration wasdriven toward a gradient of fMLP, suggesting that the proportion of PMNmigration exceeding that of fMLP controls is dependent on aLXA₄-mediated event. Moreover, these results suggest that the action ofLXA₄ may be synergistic with fMLP, since LXA₄, by itself, does notpromote PMN migration in the absence of fMLP.

In addition, it was discovered that pre-exposure of PMN to LXA₄modulates migration of PMN in a polarized manner. That is, oppositeeffects were observed depending on the direction of PMN migration. Theobserved effect of LXA₄ inhibition of PMN migration in thephysiologically oriented (basolateral-to-apical) direction was dependenton concentration- as well as the duration of pre-exposure. These effectswere found to be selective for LXA₄, since no effect was observed withthe positional isomer LXB₄.

As described for leukocyte movement across endothelia, (Butcher E. C.(1991) Cell 67: 1033-1036), PMN migration across epithelial monolayersis likely a multi-step process requiring engagement and disengagement ofseveral receptor-ligand complexes between PMN and epithelial cell. Thespecific events involved in PMN transepithelial migration are poorlyunderstood at the present time, but in part requires the PMN B2 integrinCD11b/CD18 and is independent of ICAM-1. (Parkos C. A., et al. (1991).J. Clin. Invest. 88:1605-1612). In addition, PMN transepithelialmigration can be regulated by exposure of T84 epithelial monolayers tothe lymphokine interferon-gamma (IFN-γ). In light of the polarizednature of this epithelium, (Madara, J. L., and K. Dharmsathaphorn(1985). J. Cell Biol. 101: 2124-2133), (Madara J. L., et al. (1987).Gastroenterology 92: 1133-1145), (Dharnsathaphom K., and J. L. Madara(1990). Meth. Enzymol. 192: 354-389), it would not be surprising thatthe sequence by which PMN encounters epithelial ligands directlyregulates PMN migration.

Moreover, PMN migration across endothelia requires a sequential seriesof activation and deactivation steps on the PMN surface, of whichlipid-derived activating factors may play an important role (reviewed inButcher E. C. (1991) Cell 67: 1033-1036). Whether LXA₄ could act as alipid-derived factor for expression of a crucial ligand in theregulation of PMN migration across epithelia is not known. Evidence tosupport this hypothesis are provided by a recent study characterizinglipoxin binding sites on human PMN. (Palmblad J., et al. (1987) BiochemBiophys. Res. Commun. 145:168-175). With a reported K_(d) of 0.5 nM andapproximately 1800 binding sites/cell, the range of LXA₄ concentrationsused in the present study (0.01-10 nM) should provide maximal activationof subsequent signal transduction steps, most of which remain to beelucidated, but appears to involve a G-protein associated activationstep (Fiore S., et al. (1992) J. Biol Chem. 267: 16168-16176), andpossibly a signalling step through PKC as determined by inhibition usingstaurosporine.

Recent in vivo studies have shown that LXA₄ is an importantlipid-derived mediator at several distinct anatomic sites, including thelung, (Christie P. E., et al. (1992) Am. Rev. Respir. Dis. 145:1281-1284), kidney, (Katoh T., et al. (1992) Am. J. Physiol. 263:F436-F442), blood vessel (Brezinski D. A., et al. (1992) Circulation.86: 56-63), and hamster cheek pouch (Hedqvist P., J. et al. (1989).Acta. Physiol. Scand. 137: 571-572). The data reported here suggest thatLXA₄-elicited alterations exert effects at the level of PMN andsubsequently modulate PMN-epithelial interactions. Previous studies haveshown that production of lipoxygenase products of arachidonic acidcorrelate with intestinal inflammation. Specifically, an enhancedconversion of arachidonate to 5-, 12- and 15-hydroxy-eicosatetraenoicacid (HETE) has been shown in ulcerative colitis homogenates(Broughton-Smith, N. K., et al. (1983) Gut 24: 1176-1182), as well asincreased biosynthesis of LTB₄ in Crohn's disease (Sharon, P. and W. F.Stenson (1984) Gastroenterology 86: 453-460). The present resultsdemonstrate that lipoxins, and especially by their action on PMN, play arole in intestinal disease.

EXAMPLE 2 Measurement of Electrical Parameters of Cultured EpithelialMonolayers: Use in Assessing Neutrophil-Epithelial InteractionsMATERIALS AND METHODS

General information regarding T84 cells: T84 cells were negative formycoplasma as tested commercially using a nucleic acid probe (OrganonTeKnika Corp., Rockville, Md.). T84 cells were first obtained fromDharmsathaphorn in 1984 (Dharmsathaphorn, K. Am. J. Physiol. 246(Gastrointest. Liver Physiol. 9):G204-G208; 1984). T84 cells wereoriginally isolated from a lung metastasis of a patient with coloniccarcinoma and were established as a transplantable line in BALB/c nudemice (Dharmsathaphorn, K. Am. J. Physiol. 246 (Gastrointest. LiverPhysiol. 9):G204-G208; 1984). However, T84 cells are now available fromthe ATCC (American Type Culture Collection, Rockville Md., cat #CCL248). ATCC derived T84 cells passages 60-100 have been compared with theoriginal distributed parent line (passages 16-40) by assayingelectrogenic Cl⁻ secretory responses to cAMP (8-bromo-cAMP,theophylline, or forskolin) and Ca⁺² (ionomycin, A23187, carbachol)agonists, cell migration patterns, neutrophil transmigration, andcytoskeletal responses to bacterial (C. difficile toxin A) and fingal(cytochalasin D) derived toxins. The cells from these two sources arehighly comparable. Some differences between T84 cells from these twosources do exist in conditions for loading cells with reagents whichcross the plasma membrane passively (such as loading phalloidin intoliving cells (Shapiro, M.et al; J. Clin. Invest. 87:1905-1909; 1991)).However, even within cells from ATCC, optimal loading conditions canvary substantially over 20-25 passages and thus loading conditions withcells from any source must be empirically defined in each laboratory.

Growth and Maintenance of T84 cells: As previously detailed(Dharmsathaphorn, K.; Madara, J. L. Meth. Enzymol. 192:354-389; 1990),T84 cells are grown as monolayers in a 1:1 mixture of Dulbecco-Vogtmodified Eagle's (DME) medium and Ham's F-12 medium supplemented with 15mM Na+-HEPES buffer, pH 7.5, 1.2 g/l NaHCO₃, 40 mg/liter penicillin, 8mg/liter ampicillin, 90 mg/liter streptomycin, and 5% newborn calfserum. Growth requirements are not strict as cells will grow in avariety of media (Dharmsathaphorn and Madara, personal observations,(Dharmsathaphorn, K.; Madara, J. L. Meth. Enzymol. 192:354-389; 1990).However, some care must be taken in selecting media, since mediaselected strictly for the ability to increase growth rate can result inthe loss of the polarized phenotype (Madara, personal observations).Cells are split near confluency by incubating in 0.1% trypsin and 0.9 mMEDTA in Ca+2 and Mg+2 free phosphate buffered saline for 15-20 min. T84cells grow best when split 1:2 and plating at lower densities maygreatly retard growth. After splitting, cells are generally nearconfluency once again in 5-8 days; thus they are relatively slow growingcells. T84 cells aggregate in suspension and attempts to produce uniformdispersal of cells will result in substantial loss of viability andresulting low effective plating density.

Preparation of monolayers: Normal or inverted monolayers can beconstructed for a physiological microassay using the commerciallyavailable insert system (Costar inserts, 0.33 cm², 5 μm polycarbonatefilters). The larger pore size is crucial for allowing neutrophils topenetrate the filter. Collagen 1 must coat the filters to allowattachment of T84 cells. Original descriptions of the collagen coatinvolved procedures in which the collagen was chemically crosslinkedDharmsathaphom, K. et al; Meth. Enzymol. 192:354-389; 1990.Dharmsathaphorn, K. et al; Am J. Physiol. 246 (Gastrointest. LiverPhysiol. 9):G204-G208; 1984. However, neutrophil movement acrosscrosslinked collagen gels has been found to be extremely limited.

To circumvent this problem, Cereijido and Sabatini's method (Cereijido,M.; Sabatini, D. D. 1978 J. Cell Biol. 77:853-876;) of preparing viscouscollagen from rat tail tendons is followed and this solution is mixed1:3 with 60% ethanol at 4° C. Very little collagen is required; indeedcollagen ethanol ratios of 1: 100 can be readily used. Fifty microlitersof this mixture is then placed on each filter, taking care of an evendistribution and plates are allowed to dry in a hood (3 hr—overnight). Afew drops of media are then added to the wells for 1-3 hr, and cells(10⁶/cm²) are then added in a total volume of 167 μl. Eight-hundredmicroliters of media are added to the outer wells and a few drops ofadditional fresh media to the inner well.

After reaching the initial steady state resistance, the monolayersshould be used within 6-14 days for two reasons: first, physiologicalresponses such as Cl⁻ secretion will diminish with time and second,without underlying crosslinked collagen, cell processes can eventuallymove through the 5 μM pores and ultimately result in a near doublemonolayer (monolayer on each side of the filter). Monolayers need onlyhave one feeding, but this should take place at least 24 hrs prior toexperimental use.

Inverted monolayers can also be grown using this technique. For this 0.8mm thick lexan rings having the same dimension of the base of Costarinserts are machined, deburred, cleaned by boiling with a trace ofdetergent and subsequently exhaustively washed, and attached to theunderside of the insert using General Electric RTV Silicone glue (thisunderside ring is necessary for a peripheral electrical seal). Afterdrying overnight, the inserts are sterilized by submersion in 70%ethanol (4-hr overnight), inverted onto a sterile petri dish in a hood,and allowed to dry. Collagen and cells are added to the filter(underside now facing up) exactly as with unmodified inserts and cellsare allowed to attach for 4 hours before righting the inserts into the24 well holding plates. Subsequent treatment of the monolayers isidentical to that described above.

Isolation of neutrophils: Neutrophils are isolated from whole bloodusing a gelatin-sedimentation technique. Briefly, whole blood,anticoagulated with citrate/dextrose, is centrifuged at 300×g for 20minutes (20° C.). The plasma and buffy coat are carefully removed usinga curved, siliconized glass pipette which is attached to a vacuum trap.Care must be taken not to aspirate the interface between the buffy coatand RBC since this is where PMN reside. To eliminate contaminating RBC,a 2% solution of gelatin (100 bloom, Fisher) made up in either saline orHBSS (35-40° C.) is added to the RBC/PMN mixture at a ratio of 35 ml ofgelatin per 15-20 ml of cells.

The gelatin/cell mixture is then incubated at 37° C. for 30 minutes tosettle-out contaminating RBC. The pink supernatant is then centrifugedat 400×g for 10 minutes (20° C.) to yield a red pellet of PMN and someRBC's. Residual RBC's are then lysed by gentle resuspension of thepellet with isotonic ammonium chloride (t=4° C.), followed immediatelyby centrifugation at 300×g, 10 minutes, 4° C. After washing twice inHBSS (HBSS without Ca¹⁺² or Mg⁺²), the cells can be counted andresuspended at 5×10⁷ PMN/ml and are ready for use. The above methodusually yields 1-2×10⁸ PMN from 100 ml of whole human blood at a purityof approximately 90%.

Physiological assays: All solutions and materials are maintained at 37°C. A convenient way to do this is to perform the experiments in a 37° C.room. T84 cells do well in this environment in bicarbonate free buffersfor at least four hours. Hanks Balanced Salt Solution (HBSS, Sigma,without bicarbonate or phenol red) to which 10 mM HEPES buffer is added,pH 7.4 is used for these assays. Inserts with attached monolayers arelifted from wells, drained of media by inverting, and gently rinsed bydipping in a 200 ml container of HBSS. Inserts are then placed in newwells with 800 μl fMLP in the lower compartment and subsequently 100 μlHBSS is added to the inner well (an additional 100 μl containingneutrophils in HBSS is added to the inner well to initiate theexperiment). The above treatment has little effect on resistance butwashing or other trauma does consistently result in a transienttransepithelial current (4-15 uA/cm2) which returns to baseline (0-3μA/cm²) within 4 minutes.

To measure currents, transepithelial potentials, and resistance, thefollowing system was utilized: a commercial voltage clamp (Iowa DualVoltage Clamps, Bioengineering, University of Iowa), interfaced with anequilibrated pair of calomel electrodes submerged in saturated KCl andwith a pair of Ag—AgCl electrodes submerged in HBSS. Agar bridges arethen made: HBSS containing 6% agar is heated in a water bath until theagar is in solution and the solution is perfectly clear. Using a syringefor suction, this hot solution is then pulled through 1 mm borepolyethylene tubing (12 cm lengths), the agar is allowed to gel, and theends are trimmed to a 45 tapper with a razor blade. Agar bridges arethen used to interface the electrodes with the solutions on either sideof the monolayers (one calomel and one Ag—AgCl electrode in each well).

The agar bridge pair to the inner well is properly positioned when thesurface tension of the fluid above the monolayer is broken. The pair ofbridges in the outer well is positioned by inserting the agar bridges tothe bottom of the well (through one of the openings on the side of theinsert) and then withdrawing it 1 mm. In practice, a hand-heldpolycarbonate strip, which fixes all distances and positions, had beenmade. Such a bridge-holding device makes use of the fact that alldistances are fixed (from top of well to monolayer, from side of well toinsert center, etc.). In high resistance monolayers, such as T84,positional effects are minimal. This is likely due to the relativelyhigh resistance of the monolayer which promotes relatively uniformcurrent densities at the monolayer surface. The taper in the tips of theagar bridges is also advantageous in preventing entrapment of airbubbles when gently positioning the inner bridges and in preventingabutment of the outer bridges with the plate bottom which leads tospuriously high resistance readings.

For measurements, bridges are positioned as described, and thespontaneous transepithelial electrical potential and the instantaneouspotential generated by passing 25 μA of current are measured. Similarmeasurements are taken after scraping the filter with a pipette andthese later measurements are used to correct for system resistance.Using these values and Ohm's Law, tissue resistance and transepithelialcurrent are then calculated. System resistance is less than 5% of thetotal resistance value. In a single monolayer in which the bridges weresimply held by hand and repositioned 20 times (making sure that bothelectrode pairs were varying in position) resistance and spontaneoustmnsepithelial electrical potential varied by less than 10%. In settingup inverted monolayers for study one must be cautious that small airbubbles are not trapped under the monolayer by the added ring. Thisproblem can be circumvented by lowering the washed insert into the HBSSof the outer well slowly and at an angle.

In practice the above approach allows one to serially record plates ofmonolayers and accurate readings from 36 monolayers can be obtained inless than 10 minutes.

Myeloperoxidase assay of neutrophil transepithelial migration: PMNcontents of monolayers and lower chambers can be quantitated by assayingthe PMN-specific azurophil granule marker, myeloperoxidase (MPO).Monolayers are cooled to 4° C. and washed with HBSS using a pipette toremove non-adherent PMN. Washed monolayers are then placed in new24-well tissue plates and overlaid with 1.0 ml 0.5% Triton X-100 in HBSSin order to solubilize PMN-associated MPO. After 10 minutes of vigorousshaking, monolayers can then be discarded and supernatant saved forassay. To the original lower chambers, MPO can be solubilized by simplyadding 50 μl of 10% Triton X-100 and mixing.

To assay for solubilized MPO, the pH of solubilized chambers andmonolayers must be adjusted to 4.2 using 100 μl of 1.0 M citrate pH 4.2.Aliquots of each pH adjusted sample can then be transferred to a 96-wellmicrotiter plate for substrate addition. After adding 100 ul of asolution of 2 mM 2,2′-azino-di-(3-ethyl) dithiazoline sulfonic acid(ABTS), 0.06% H₂O₂ in 100 mM citrate buffer pH 4.2 to each well, colordevelopment can be quantitated on a microtiter plate reader at 405 nm.The reaction can be terminated by the addition of sodium dodecyl sulfateto a final concentration of 0.5%. For standards, serial dilutions of thesame PMN used in the experiment can be made in 1.0 ml of HBSS. MPO issolubilized identically as for the lower chambers. When performed inthis manner, the assay is linear in the range of 0.3-50×10⁴ cells/ml.

RESULTS

Steady state resistance was reached within 7 days of plating and valuesgreater than 1,000 ohm cm² were achieved. Both the time course and valueof resistance are comparable to that achieved on matrices ofcross-linked collagen (Omann, G. M. et al.; Physiol. Rev. 67:285-322;1991). Passage-related variation in time to reach physiologic confluence(4-7 days) and in resistance value in the steady state (500-1900 ohmcm²) does occur. Resistance is measured by the simple bridge methoddescribed. The time course and steady state resistance value achieved iscomparable to that previously published for monolayers on thickcross-linked collagen I matrices measured by formal Ussing chamber means(Madara, J. L; Dharmsathaphom, K. J. Cell Biol. 101:2124-2133; 1987).The collagen matrix does not inhibit epithelial transmigration byneutrophils and thus is suitable for such assays. The approach tomeasurement of resistance allows one to obtain sequential values fromnumerous monolayers over a short duration of time.

Neutrophil migration across T84 monolayers was accompanied by asignificant decrease in transepithelial resistance when migration was inthe apical to basolateral direction. The size of the resistance decreasewas paralleled by the density of applied neutrophils. Neutrophildensities in cell number/cm² are indicated. Transmigration (apical tobasolateral) was stimulated by a 10⁻⁷ M transepithelial gradient offMLP. The decrease in resistance due to penetration of intracellulartight junctions by neutrophils was large at the highest neutrophildensity and is saturated within 60 minutes. Furthermore, as indicated bythe myeloperoxidase assay, the log of resistance correlates well withthe number of neutrophils migrating into the membrane (cells havingcrossed the tight junction but remaining above the filter) and with thetotal number of transmigrated cells. The log of the final resistancevalue (5×10⁶ neutrophils/cm² in 10⁻⁷ M fMLP gradient for 110 min)correlates with the number of neutrophils transmigrated. Variations inelectrical responses are due to variations in efficacy oftransmigration.

The decrease in resistance was only modest in the absence of a gradientand under these conditions the number of transmigrating neutrophils wassmall. Antibody to the common beta chain of neutrophil β₂ integrins(CD18) blocks transmigration and the fall in resistance while a controlantibody (J5) recognizing CD10 has no inhibitory effect. The fall inresistance seen in controls (no fMLP, no PMN) and in the presence ofanti-CD18 antibody represents, in large part, the 200-300 ohm fall inresistance occurs when monolayers are transferred from media to HBSS.

fMLP was effective in stimulating transmigration of neutrophils at both10⁻⁶ and 10⁻⁷ M as measured by either resistance or myeloperoxidaseassay.

TABLE 1 NEUTROPHILS MIGRATE ACROSS INVERTED, SPARSELY COLLAGEN-COATEDINSERTS AND SUBSEQUENTLY ACROSS T84 MONOLAYERS (i.e. BASOLATERAL TOAPICAL) Neutrophil Cell Equivalents × 10⁴* In Opposite In MonolayersReservoir Total No FMLP⁺ 0.23 0.18 0.41 No PMN 0 0 0 FMLP + PMN 5.4785.30^(#) 90.77^(#) FMLP + PMN + αCD18 1.22 5.76 7.00 *Values representmeans of three determinations; ⁺FMLP = 10⁻⁶M; PMN = 6 × 10⁶/cm²;^(#)FMLP + PMN are significantly different (P < .0001) from all othergroups.

Additionally, the blocking antibody to CD18 inhibited neutrophiltransmigration nearly completely as assessed by each assay, whilesubstantial transmigration proceeded in the presence of control antibody(anti-CD10). Transmigration which is inhibitable by antibody to CD18also occurred in the basolateral to apical direction (Table 1).Neutrophils applied to the basolateral surface of monolayers haveeffects on resistance independent of transmigration and thus themyeloperoxidase assay is most useful in this direction as a quantitativemeasure of transmigration.

As previously reported in Nash, S. et al; J. Clin. Invest. 87:1474-1477;1991, a neutrophil derived secretagogue (NDS) activity, detected as amodest short circuit current, occurs as a consequence of neutrophil-T84cell interactions. Such currents are readily detected in the microassaydescribed, are important since they represent active electrogenictransepithelial transport events, and are not readily detected using“chop-stick” type detection systems.

DISCUSSION

Detailed here are useful approaches in utilizing T84 cells for studiesof epithelial monolayer finction in general and epithelial-neutrophilinteractions in particular. Electrical data obtained from Ussingchambers designed for cultured monolayers (Dharmsathaphom, K.; Madara,J. L., Meth. Enzymol. 192:354-389; 1990. Nash, S. et al; J. Clin.Invest. 87:1474-1477; 1991. Dharmsathaphorn, K. et al; Am. J. Physiol.246 (Gastrointest. Liver Physiol. 9):G204-G208; 1984) are highlyaccurate. However, in using such systems one is limited by the number ofsuch chambers available, each of which is occupied by one monolayer forthe length of the experiment. Additionally, such systems havetraditionally been designed to accommodate monolayers with large surfaceareas (such as 2 cm²) since larger surface areas provide for moreaccurate transepithelial flux measurements. However, flux measurementsare often only required to address specific issues and the bulk of manyexperiments can often be completed with simple electrical assays ofresistance and short circuit current.

Electrical data with reproducibility rivaling that of more formalizedbut tedious systems can be obtained, in high resistance epithelialmonolayers such as T84, by simply interfacing electrical measuringsystems usually utilized in more formal Ussing chamber systems, with acommercially available insert system. It would not be surprising if theelectrical data derived from this system were of less quality in lowresistance monolayers, since imposed current densities at the monolayersurface would likely be less uniform and therefore positional effectscould be more problematic. Moreover, it is not likely that the insertsystem will be suitable for non-electrical assays of permeability suchas flux. Major limitations in this regard are the lack of stirring andthe overly small compartment (apical) from which to obtain samples.

Also described is a simple procedure for manufacturing invertedmonolayers so that neutrophils or other cells can settle by gravity onthe basolateral side of the monolayer. In this system, attention tocomposition of the matrix on which one plates cells determine whethercells and macromolecules added to the basolateral surface are able togain access to the basolateral membrane of the epithelial cell. Lastly,a simple enzymatic assay which allows quantitation of numbers ofneutrophils which have migrated either into or across epithelialmonolayers is described.

The data presented, which show examples of the application of theseapproaches to types of studies previously assessed in formalized Ussingchamber experiments, indicate that such methods are useful for detectingthe physiologic effects of neutrophil-epithelial interactions onepithelial barrier function and activated transepithelial electrogenictransport. The approach outlined can be used to show that intestinalepithelial barrier function is diminished and Cl⁻ secretion is activatedby such neutrophil-epithelial interactions. Both of these functionaldisorders of intestinal epithelia are known to occur in vivo in humansaffected by intestinal diseases characterized by neutrophil infiltrationof the epithelium. Thus, such in vitro cell culture models of theinteractions between these two cell types provide an opportunity formechanistic studies of the symptomatology occurring in these importanthuman diseases,

EXAMPLE 3 Synthesis of Lipoxin Analog Compounds

Preparation of the Methyl Ester Precursor of Compound 1

To a solution of 3-methyl-3-trimethylsiloxy-1-bromo-1-octene (130 mg.0.44 mmol) in benzene (1.5 mL) was added n-propylamine (0.05 mL, 0.61mmol) and Pd(PPh₃)₄ (20 mg. 0.02 mmol) and the solution was protectedfrom light. It was then degassed by the freeze-thaw method and stirredat rt for 45 min. (7E, 9E, 5S, 6R) Methyl5,6-di(tert-butyldimethylsiloxy)-dodeca-7,9-diene-11-ynoate (183 mg.0.44 mmol) (compound 12) and copper iodide (14 mg. 0.07 mmol) were addedand the solution was one more time degassed by the freeze-thaw method.The mixture was stirred for 3 h at rt and quenched with saturatedaqueous solution of NH₄Cl and extracted with ether. It was then washedwith brine and dried over MgSO₄ and the solvent was evaporated. Flashcolumn chromatography (silica, 3% ether hexanes) afforded pure compoundas a colorless liquid (171 mg. 57% yield).

To a solution of the compound (171 mg. 0.25 mmol) in THF (0.5 mL) wasadded n-BuN₄F(0.9 mL. 0.90 mmol) and the mixture was stirred at rt. Thereaction was completed in 2 h at which time it was poured into water andextracted with ether. The ether extracts were washed with brine, driedover Na₂SO₄ and the solvent was evaporated. Flash column chromatography(silica 4% MeOH/CH₂Cl₂) afforded the methyl ester (24 mg.) together withsome of the corresponding lactone. HPLC retention time: 9:39 min(microsorb reverse phase, 4.6 mm×25 cm, C-18 column, MeOH/H₂O 70:30 flowrate 1 ml/min, V detector at 300 nm). UV in MeOH: λ _(max)283, 294, 311nm. ¹H NMR (500 MHz CDCl₃) δ6.53 (dd. 15.2 10.9 Hz, 1 H), 6.32 (dd,J=15.1, 11.0 Hz, 1 H), 6.17 (d, J=15.9 Hz, 1 H) 5.83 (dd. J=17.5, 2.1Hz, 1 H), 5.80 (dd. J=15.2, 6.7 Hz, 1 H), 5.72 (dd. J=17.0, 2.1 Hz, 1H), 4.14 (m, 1 H), 3.68-3.64 (m, 4H), 2.35-2.31 (m, 2 H), 1.51-1.48 (m,1 H), 1.43-1.42 (m, 2 H), 1.30-1.23 (m, 15H) 0.85(t,3 H). ¹³C NMR (126MHz, CDCl₃) δ 150.01, 140.18, 132.95, 132.26, 112.43, 107.50, 75.23,73.76, 42.49, 33.67, 32.17, 31.36, 27.96, 23.56, 22.58, 21.03, 14.03.

Preparation of the Methyl Ester Precursor of Compound 2

A solution of the methyl ester precursor of compound 1 (3 mg. in CH₂Cl₂(1 ml) was mixed with Lindlar's catalyst (1 mg.) and placed under ahydrogen atmosphere. The mixture was stirred at rt in the dark followedby HPLC until about 80% conversion (1 h). Filtration over celiteevaporation of the solvent and separation by HPLC gave a pure methylester. HPLC retention time: 10:02 min (microsorb reverse phase. 10 mm×25cm C-18 column, MeOH/H₂O 70:30 flow rate 4 ml/min. UV detector at 300nm). UV in MeOH: η_(max) 287, 301, 315 nm.

Preparation of the Methyl Ester Precursor of Compound 3

This compound was prepared similarly to the preparation of the methylester precursor of compound 1 (from3-cyclohexyl-3-trimethylsiloxy-1-bromo-1-octene). Desilylation of thiscompound was also performed in a similar manner to afford the methylester. HPLC retention time 8:02 min (microsorb reverse phase, 4.6 mm×25cm. C-18 column, MeOH/H₂O 70:30, flow rate 1 ml/min, UV detector at 300nm). UV in MeOH: λ_(max) 282, 293, 311 nm. ¹H NMR (360 MHz, CDCl₃) δ6.56(dd, 15.4, 10.9 Hz, 1 H), 6.33 (dd, J=15.2, 10.9 Hz, 1 H), 6.13 (dd,J=15.8, 6.5 Hz, 1 H), 5.81 (dd, J=15.2, 6.4 Hz, 1 H), 5.80 (d, J=15.6Hz, 1 H), 5.73 (dd, J=15.4, 2.1 Hz, 1 H), 4.15 (br, 1 H), 3.93-3.90 (m,1 H), 3.67 (br, 1 H), 3.65 (s, 3 H), 2.34 (t, 2 H), 1.82-1.65 (m, 10 H),1.46-1.38 (m, 3 H), 1.26-1.01 (m, 5 H).

Preparation of the Methyl Ester Precursor of Compound 4

Selective hydrogenation of the methyl ester precursor of compound 3,followed by HPLC purification gave the methyl ester precursor ofcompound 4. HPLC retention time: 9.72 min (microsorb reverse phase, 10mm×25 cm C-18 column, MeOH/H₂O 70:30 flow rate 4 ml./min. UV detector at300 nm), UV in MeOH: λ_(max) 288, 301, 315 nm. ¹H NMR (250 MHz, C₆D₆) δ6.66-6.89 (m, 2 H), 5.95-6.24 (m, 4 H), 5.55-5.66 (m, 2 H), 3.82 (m, 1H), 3.73 (m, 1 H), 3.41 (m, 1 H), 3.31 (s, 3H, OCH₃), 2.08 (t, 2 H,CH₂COO), 1.00-1.81 (m, 18 H).

The methylesters can be converted to corresponding alcohols usingstandard techniques.

Synthesis of 15(R)-15-methyl-LXA₄ and 15(±)methyl-LXA₄

Approximately 1 gm acetylenic ketone a is prepared using Friedel-Craftsacylation of bis(trimethylsilyl) acetylene with hexanoyl chloride and isreduced using (−)-pinayl-9-BBN to give the (S) alcohol in CH₃N₂ as inWebber, S. E. et al. (1988) Adv. Exp. Med. Biol. 229:61; Nicolaou, K. C.et al. (1991) Angew. Chem. Int. Ed. Engl. 30:1100; and Vorbr{umlaut over(u)}ggen, H. et al.: In: Chemistry, Biochemistry, and PharmacologicalActivity of Prostanoids (Roberts, S. M., Scheinmann, F. eds.). Oxford:Pergamon Press, to generate the methyl at C-15.

Alternatively, the keto group can be treated with CH₃MgBr (60→70° C.) asin Vorbr{umlaut over (u)}ggen, H. et al.: In: Chemistry, Biochemistry,and Pharmacological Activity of Prostanoids (Roberts, S. M., Scheinmann,F. eds.). Oxford: Pergamon Press to yield the 15(±)methyl of b (2-5 g)in dry CH₂Cl₂ (˜20 ml) at 0° C. with sequential additions of2,6-lutidine (5.2 ml) and tert-butyldimethylsilyl triflate (6.9 ml).This reaction is mixed for 1 h and then diluted with 100 ml ether foraqueous extraction and drying with MgSO₄.

The product c is then coupled with d

that is generated as in Nicolaou, K. C. et al. (1991) Angew Chem. Int.Ed. Engl. 30:1100; Nicolaou, K. C. et al. (1989) J. Org. Chem. 54:5527and Webber, S. E. et al. (1988) Adv. Exp. Med. Biol. 229:61. Structure dfrom fragment A in Scheme I is suspended in 4.0 equiv. of AgNO₃, then7.0 equiv. of KCN, containing EtOH:THF:H₂O (1:1:1), 0-25° C. for 2 h togenerate the C-methyl ester protected 15-methyl-LXA₄ analog that isconcentrated and saponified in THF with LiOH (2 drops, 0.1 M) at 4° C.12-24 h to give the corresponding free acid.

Synthesis of 16-dimethyl-LXA₄

This compound is generated using the similar strategy by coupling dabove with e vide supra, or f to generate the 15-phenyl-LXA₄ analog, org to generate the 17-m-chlorophenoxy-LXA₄ analogs.

The appropriate C fragments in Scheme I (i.e. e, f, g, h,) are eachprepared as reviewed in Rad{umlaut over (u)}chel, B. and Vorbr{umlautover (u)}ggen, H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res.14:263 for the known corresponding prostaglandin analogues. In h, R═H;Cl, methoxy or halogen.

Synthesis of 13,14-acetylenic-LXA₄ and Halogen-containing Analogs.

Using the A₂B₂ generated fragment from Scheme II, the corresponding C₂fragments are prepared for coupling. Structures j and k are generated asin Nicolaou, K. C. et al. (1989) J. Org. Chem. 54:5527 and methylated asin Rad{umlaut over (u)}chel, B. and Vorbr{umlaut over (u)}ggen, H.(1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14:263 arecoupled to 7 to yield these LX analogues. The materials may be subjectto RP-HPLC for purification vide supra.

Synthesis of 14,15-acetylenic-LXA₄.

The designated combined A₂B₂ fragment can be prepared from couplings offragments A₁ and B₁, illustrated in Route II to carry the structure of 7or 4 vide supra for coupling to fragment C₂. The precursor for the C₂fragment 1 can be prepared as in Rad{umlaut over (u)}chel, B. andVorbr{umlaut over (u)}ggen, H. (1985) Adv. Prostaglandin ThromboxaneLeukotriene Res. 14:263 for a prostaglandin analog.

Precursor m as prepared previously (Nicolaou, K. C. (1989) J. Org. Chem.54:5527) is added at 1.2 equiv. to 0.05 equiv. of Pd(PPh₃)₄, 0.16 equiv.of CuI, n-PrNH₂, in benzene with Me₂Al-carrying 1, 2-3 h RT to yield n.

The alcohol protecting groups TBDMS=R are removed with 10 equiv. ofHF-pyr, THF, 0-25° C. (4 h) followed by exposure to 3.0 equivalents ofEt₃N, MeOH, 25° C. 15 min to open acid-induced δ-lactones that usuallyform between C-1-carboxy and C-5 alcohol in the lipoxins (Serhan, C. N.(1990) Meth. Enzymol.187:167 and Nicolaou, K. C. (1989) J. Org. Chem.54:5527). After mild treatment with Lindlar cat. 5% by weight, theextracted material may be subjected to LiOH saponification in THF togenerate the free acid of the target molecule that can be subject tofurther purification by RP-HPLC gradient mobile phase as in (Serhan, C.N. et al. (1990) Meth. Enzymol. 187:167).

Synthesis of 15(±)methyl-cyclo-LXA₄

Compound o as the SiMe₃ derivative can be placed (˜1 gm) in a roundbottom 100 ml flask under an atmosphere enriched with argon in degassedbenzene (20 ml). To this add 3.0 equivalents of a vinyl bromide fragmentvide infra. This coupling reaction is carried out in catalytic amountsof Pd (PPh₃)₄ and CuI and can be monitored by injected aliquots of thissuspension into RP-HPLC monitored by UV abundance with a rapid scanningdiode. The progression line course 1-3 h at 23° C. after which thematerial is extracted with ethyl acetate: H₂O 4:1 v/v) and concentratedby rotoevaporation. The methyl ester can be saponified in LiOH/THF togive quantitative yields of the free carboxylic acid. Other derivativescan be prepared as above using fragment A with different fragment Bmoieties that have been substituted to give for example a dimethyl orother derivative. This can be obtained by taking the readily availableketone p and treating it with CH₃MgBr (60° C.) to generate q that canalso be coupled to fragment A as above using conventional techniquessuch as Pd(O)-Cu(I) coupling. Increased chain length from C-15 can alsobe obtained.

Synthesis of 5-Methyl-LXB₄ and 4,4-Dimethyl-LXB₄.

The 5-methyl-LXB₄ hinders or retards 5-oxo-LXB₄ formation. Using thegeneral scheme outlined above, the A fragment can be constructed tocarry the 5-methyl in a vinyl bromide r precursor that is coupled toajoined B+C fragment by Pd(O)-Cu(I) coupling.

The vinyl bromide r can be obtained from the s that contains eitherdimethyl or hydrogen substituents at its C-4 position. The protectedprecursor t containing fragments B+C is generated as reported inreference (Nicolaou K. C. et al. (1991) Angew. Chem. Int. Ed. Engl. 30:1100-16.). Compound t is converted to s or 28 by coupling with theindicated vinyl bromide. Thus the target molecule can be generated byadding rat 1.0 equv. (≈1 gm) to a round bottom flask degassed containingEt₂NH as solvent with t injected in Et₂NH at 1.2 equiv. Pd(Ph₃P)₄ isadded at 0.02 equiv. to give the 8(9)-containing acetylenic precursormethyl ester of s.

The material is extracted and subject to rotoevaporation suspended inquinoline (0.5 eq) in CH₂Cl₂ and subject to hydrogenation using (10%;25° C.) Lindlar catalyst and a stream of H₂ gas to selectively reducethe acetylenic double bond at position 8. The formation of the tetraenecomponent of the methylester of 5-methyl-LXB₄ or 4-dimethyl-LXB₄ methylester can be monitored by RP-HPLC to assess completion of the reduction(i.e., 1-3 h). The methyl#esters are next saponified to theircorresponding free acids by treating the products with LiOH in THF 25 μlH₂O added at 0→24°, 8-24 h.

EXAMPLE 4 Activity of Lipoxin Analogs on Columnar Epithelia

Several of the preferred lipoxin analogs (shown structurally ascompounds 1 through 8 in Example 3) were prepared by total synthesis asdescribed in Example 2. Following preparation and isolation of thesecompounds via HPLC, compounds were assessed to determine whether theyretain biological activity using the epithelial cell transmigrationassays as described above in Example 1.

Compounds 1 through 8 (10⁻⁷-10⁻¹⁰M) were found to inhibit neutrophiltransmigration on epithelial cells. The acetylenic precursors (compound1, 3, 5 and 7) were found to be physically more stable than theirtetraene counterparts. Compound 7, which did not have an alcohol groupin the C15 position or other modifications in the series, showed nobiological activity in the assays. It would therefore appear that asubstituent in the C15 position of lipoxin is necessary for thebiological activity of at least lipoxin A₄ analogs. Lipoxin analogs 1through 8 were found to block migration at potencies greater than orequal to synthetic lipoxin A₄. Compounds 1, 2 and 4 were found to beparticularly effective. The results indicate that lipoxin A₄ analogswith modifications in C15-C20 positions retain their biological actionand can inhibit PMN transmigration in columnar epithelia.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

We claim:
 1. A compound having the formula:


2. A compound having the formula: